Neuroscience

Cerebrospinal fluid-contacting neurons (CSF-cNs) are a specialized group of multifunctional neurons located around the central canal of the spinal cord. They play critical roles in motor regulation, postural maintenance, and spinal cord injury repair. However, the molecular mechanisms underlying the multifunctionality of CSF-cNs remain poorly understood, partly due to the lack of established in vitro methods for their efficient selection and purification, which significantly hinders mechanistic investigations. In this study, we describe a standardized method using a PKD2L1 promoter-driven lentiviral system, which enables effective enrichment and identification of CSF-cNs in vitro through GFP labeling and puromycin selection. This protocol includes key steps such as construction of the PKD2L1 promoter-driven lentiviral vector, spinal cord tissue collection and digestion from neonatal mice, lentiviral infection, antibiotic selection, and immunofluorescence-based identification of CSF-cNs. Our method provides a reliable platform for obtaining high-purity CSF-cNs (>99%), which facilitates their functional and mechanistic studies for regenerative approaches in vitro.

Nociception is critically shaped by descending modulation of spinal circuits, yet its cellular and synaptic mechanisms remain poorly defined. Elucidating these mechanisms is technically challenging, as it requires simultaneous activation of primary afferents and descending fibers while monitoring the functioning of individual spinal neurons. Here, we present a method to investigate the influence of the rostral ventromedial medulla (RVM), a principal supraspinal structure mediating descending modulation, on the activity of spinal lamina I neurons. Our approach combines electrophysiological recordings in ex vivo intact spinal cord preparation with optogenetics, granting several advantages. First, ex vivo preparation spares rostrocaudal and mediolateral spinal architecture, preserving lamina I as well as primary afferent and descending inputs. Second, virally mediated channelrhodopsin-2 (ChR2) expression enables selective photostimulation of RVM-originating fibers. When coupled with patch-clamp recordings, this photostimulation allows identifying postsynaptic inputs from RVM to spinal neurons and revealing RVM-dependent presynaptic inhibition of primary afferent inputs. Overall, our approach is well-suited for investigating both pre- and postsynaptic mechanisms of descending modulation in physiological and pathological pain conditions.

Peripheral nerve injury (PNI) is an excellent model for studying neural responses to injury and elucidating the mechanisms that can facilitate axon regeneration. As such, several animal models have been employed to study regenerative mechanisms after PNI, including Aplysia, zebrafish, rabbits, cats and rodents. This protocol describes how to perform a sciatic nerve injury and repair in mice, one of the most frequently used models to study mechanisms that facilitate recovery after PNI, and that takes advantage of the availability of many genetic models. In this protocol, we describe a method for using fibrin glue to secure the proximal and distal stumps of an injured nerve in close alignment. This method facilitates the alignment of nerve stumps, which aids in regeneration of both sensory and motor axons and allows successful reconnection with peripheral targets.

Although axons in the peripheral nervous system can regenerate, functional recovery after nerve injuries is poor. Activity-based therapies, such as exercise and electrical stimulation, enhance the regeneration of cut peripheral axons. Despite their effectiveness, clinical application of these experimental techniques has been limited. At least part of the basis for this translational barrier has been a lack of information as to the precise mechanism of activity-based therapies on peripheral axon regeneration. To evaluate the requirements for neuron-type specific activation to promote regeneration using these therapies, in the current protocol, we employed optogenetics. Utilizing the advantages of transgenic mouse lines we targeted opsin expression to different neuron types. Using fiber optics we activated those neurons with high temporal specificity as a model of activity-based intervention after nerve injury and to measure functional recovery achieved after such a treatment.

Although it is known that the generation of movements is performed to a large extent in neuronal circuits located in the spinal cord, the involved mechanisms are still unclear. The turtle as a model system for investigating spinal motor activity has advantages, which far exceeds those of model systems using other animals. The high resistance to anoxia allows for investigation of the fully developed and adult spinal circuitry, as opposed to mammals, which are sensitive to anoxia and where using neonates are often required to remedy the problems. The turtle is mechanically stable and natural sensory inputs can induce multiple complex motor behaviors, without the need for application of neurochemicals. Here, we provide a detailed protocol of how to make the adult turtle preparation, also known as the integrated preparation for electrophysiological investigation. Here, the hind-limb scratch reflex can be induced by mechanical sensory activation, while recording single cells, and the network activity, via intracellular-, extracellular- and electroneurogram recordings. The preparation was developed for the studies by Petersen et al. (2014) and Petersen and Berg (2016), and other ongoing studies.

Neuropathic pain is one of the highly debilitating chronic pain conditions, for which, currently, there is no therapeutic treatment. In order to reveal the underlying mechanism for neuropathic pain, various animal models have been established (Burma et al., 2016). This protocol describes how to prepare spinal nerve injury model (Kim and Chung, 1992; Rigaud et al., 2008; Masuda et al., 2016), one of the most frequently-used and highly reproducible models in which multiple alterations occur both in the peripheral and central nervous system.

The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin et al., 2005). Here we describe a procedure for the isolation of the brainstem-spinal cord tissue of neonatal rat (0-2 days old). A surgical method under binocular microscope allows the brainstem and the spinal cord to be isolated in vitro and the motor outputs to be recorded. This preparation can then be used for diverse experimental approaches, such as electrophysiology, pharmacology or anatomical studies, and constitutes a useful model to study the interaction between CPGs (Juvin et al., 2007; 2012; Giraudin et al., 2012; Le Gal et al., 2014; 2016).